Viruses Masquerading as Antibodies in Biosensors: The Development of the Virus BioResistor

Abstract

The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures. Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required. This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the Virus BioResistor (VBR). The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3−7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.

Enhancing the Sensitivity of the Virus BioResistor by Overoxidation: Detecting IgG Antibodies

“…The overoxidation process causes a bleaching of the blue VBR channel (Figure S3b), rendering the channel of the O 2 VBR colorless and transparent, consistent with strong de-doping of the PEDOT-PSS layer of the channel . Nyquist plots for a VBR and O 2 VBRs (Figure S3e) show a characteristic semicircular response expected for the VBR. − The resistive component of the channel impedance, Z re , is approximately equal to the low-frequency limit of these traces, which increases from 2 kΩ for the VBR (green) to 7.5 kΩ for the O 2 VBR prepared using a 50 s (red) overoxidation duration and to 16.3 kΩ for the O 2 VBR prepared using a 100 s duration of overoxidation (purple). This impedance increase is also consistent with a loss of mobile polaron and bipolaron charge carriers from the VBR channel .…”

“…It has been proposed that the virus-PEDOT layer of the VBR operates analogously to these chemiresistors with the conductive PEDOT component of the virus-PEDOT layer undergoing a decrease in volume fraction upon affinity-driven absorption of target protein molecules by the entrained virus particles and concomitant swelling of the virus-PEDOT layer . The resulting increase in the electrical impedance of the VBR is correlated with the concentration of the target protein over a concentration range spanning 2–4 orders of magnitude. − …”

“…VBRs and O 2 VBRs share the same device architecture consisting of a two-layer polymeric channel that is deposited on top of two gold electrodes spaced by 1.5 mm. The topmost PEDOT polymer layer also contains virus particles that are engineered using the technique of phage display to recognize and bind a particular protein. − The gold electrodes measure the electrical impedance of the polymeric channel, which increases when the embedded virus particles bind to their target protein.…”

“…Overoxidation alters the topography and thickness of the virus-PEDOT layer exposed at the surface of the VBR channel. Plan-view SEM images of VBRs and O 2 VBRs (shown in Figure a) are indistinguishable, however. − In these images, M13 virus particles and aggregates of particles appear black because they are electrical insulators and PEDOT, a conductor, appears gray. Regions of high M13 density and low M13 density are thus readily identified.…”

“…Biosensors capable of rapid, sensitive quantitation of protein disease markers in a variety of bodily fluids at the point of care could aid in the diagnosis of disease and improve prognoses. − The Virus BioResistor or VBR is a biosensor that exploits engineered virus particles (Figure S1) as receptors to detect proteins. The device’s simple chemiresistor architecture consists of embedded virus particles in a two-layer polymeric channel consisting of a conductive poly­(3,4-ethylenedioxythiophene)-poly­(styrene sulfonate) or PEDOT-PSS bottom layer prepared by spin coating and an electrodeposited virus-PEDOT top sensing layer. − We recently demonstrated that VBRs can be used to detect the bladder cancer marker DJ-1 (MW of 22 kDa) with a limit of detection, LOD DJ‑1 , of 10 pM in human urine. , However, the VBR is less sensitive to a larger protein, human serum albumin (HSA, 66 kDa), where an LOD HSA of 7.5 nM has been achieved. − The reduced sensitivity of the VBR can be traced to the reduction of the signal, ΔR VBR , for measurements of HSA compared to DJ-1 (Figure ). This depressed sensitivity of HSA relative to DJ-1 is observed across the entire VBR dynamic range for the detection of these two proteins.…”